The present invention relates to gene synthesizers and in particular to a general purpose, programmable oligonucleotide sequence synthesizer, operable in response to a pre-programmed linking chemistry and an operator selected nucleotide sequence.
Along with the evolution of an understanding of the function, structure and chemical makeup of nucleotide sequences, such as DNA, so too has an awareness evolved as to the practicalities and feasibilities of genetic engineering. Such engineering efforts, however, require a complete understanding of the chemical and biological reactions in cells. While recent efforts have demonstrated that desired bacterial mutations having desired properties can be engineered, such efforts have been very costly in terms of time, money and equipment.
The most practical of such cellular or genetic engineering efforts, to date, have been achieved via a building block approach that starts with the identification of a desired sequence of nucleotides and progresses to the growth of the desired nucleotide suquences from the component bases; to the modification of identified DNA molecules with the grown sequences; and finally to the seeding of host bacteria with the modified DNA so as to cause the production of the modified DNA via the reproduction of the cells having the sought-after modified DNA characteristics. The success of such processes thus require the linking of a desired nucleotide sequences with appropriately fragmented DNA sequences at desired sites so as to modify the DNA. The modified DNA is then introduced and reproduced by host cells, that are chemically tricked into accepting and reproducing the modified DNA. Such reproduction may also be enhanced via various so called "promotors" which are added to the modified gene. Sufficient quantities of the thus modified genes or protein are then grown via apparatus that promotes the growth of the seeded hosts and at the same time the modified DNA so as to replicate vast numbers of the seeded host bacteria and thereby the modified DNA.
Basic to such genetic engineering efforts is the synthesis of desired nucleotide chains from an initial mononucleotide. In this regard, electro-mechanical apparatus has been developed for synthesizing desired oligonucleotide sequences via the sequential linking of desired bases to a starting or "seed nucleotide." For example, various synthesizing apparatus has been developed by a number of companies, such as the Model 280 synthesizer by Vega Biochemicals, the Model 380A by Applied Biosystems and the DNA/RNA synthesizer developed by the Biologicals Company. The details of such apparatus can be obtained upon reference to the sales literature and various other published literature from these companies as well as upon reference to an article in High Technology, Volume I, No. 1 pp. 60-68 (September/October 1981).
While the above synthesizers offer alternative methods for synthesizing desired nucleotide suquences, each suffers from various shortcomings, such as excessive reagent waste or programming limitations as to the type of chemistry that can be employed. Furthermore, such synthesizers are relatively difficult to operate, maintain and refill with the necessary base/reagent/solvent materials.
The present invention, however, overcomes the above problems and incorporates the above desired features in a general purpose, programmable, micro-processor controlled synthesizer. In particular, the present apparatus is expandable to perform any of the three basic, generally accepted chemistries (i.e. phosphoramidite, phosphate triester or phosphite triester), although the embodiment disclosed hereinafter is of the phosphate type. (A more detailed description of such chemistries can also be found among other places in Nucleic Acids Research Volume 10, No. 5, pp. 1755-1769 (1982) and Hendrickson et al., Organic Chemistry, Chptr. 25-5, pp. 1007-1011 (1970)). Thus, depending upon the selected chemistry and its associated sequence of subroutines, the present apparatus successively meters programmed volumes of the process dependent base/reagent/solvents into a reation cell, and there the desired nucleotide sequences are grown in a liquid suspension on a solidsupport material. While the chemical process may be varied, the present apparatus generally operates to sequentially wash and dry the contents of the cell, expose desired nucleotide reaction sites, add and couple desired bases at the reaction sites and cap or protect the reaction sites, until the next base addition. This process then continues until a desired growth sequence is complete and after which the grown oligonulceotide is chemically separated from the solid support material.
The above objects, advantages and distinctions of the present invention as well as various others will, however, become more apparent upon a reading of the following description and upon reference to the following drawings. It is to be recognized though that while the following description is made with respect to the presently preferred embodiment, various changes (and some of which will be mentioned hereinafter) may be made thereto without departing from the spirit and scope of the present invention.